Fed-batch cultures of Hansenula polymorpha were studied to develop an efficient biosystem to produce recombinant human serum albumin (HSA). To comply with this purpose, we used a high-purity oxygen-supplying strategy to increase the viable cell density in a bioreactor and enhance the production of t...
Fed-batch cultures of Hansenula polymorpha were studied to develop an efficient biosystem to produce recombinant human serum albumin (HSA). To comply with this purpose, we used a high-purity oxygen-supplying strategy to increase the viable cell density in a bioreactor and enhance the production of target protein. A mutant strain, H. polymorpha GOT7, was utilized in this study as a host strain in both 5-l and 30-l scale fermentors. To supply high-purity oxygen into a bioreactor, nearly 100% high-purity oxygen from a commercial bomb or higher than 93% oxygen available in situ from a pressure swing adsorption (PSA) oxygen generator was employed. Under the optimal fermentation of H. polymorpha with highpurity oxygen, the final cell densities and produced HSA concentrations were 24.6 g/l and 5.1 g/l in the 5-l fermentor, and 24.8 g/l and 4.5 g/l in the 30-l fermentor, respectively. These were about 2-10 times higher than those obtained in air-based fed-batch fermentations. The discrepancies between the 5-l and 30-l fermentors with air supply were presumably due to the higher contribution of surface aeration over submerged aeration in the 5-l fermentor. This study, therefore, proved the positive effect of high-purity oxygen in enhancing viable cell density as well as target recombinant protein production in microbial fermentations.
Fed-batch cultures of Hansenula polymorpha were studied to develop an efficient biosystem to produce recombinant human serum albumin (HSA). To comply with this purpose, we used a high-purity oxygen-supplying strategy to increase the viable cell density in a bioreactor and enhance the production of target protein. A mutant strain, H. polymorpha GOT7, was utilized in this study as a host strain in both 5-l and 30-l scale fermentors. To supply high-purity oxygen into a bioreactor, nearly 100% high-purity oxygen from a commercial bomb or higher than 93% oxygen available in situ from a pressure swing adsorption (PSA) oxygen generator was employed. Under the optimal fermentation of H. polymorpha with highpurity oxygen, the final cell densities and produced HSA concentrations were 24.6 g/l and 5.1 g/l in the 5-l fermentor, and 24.8 g/l and 4.5 g/l in the 30-l fermentor, respectively. These were about 2-10 times higher than those obtained in air-based fed-batch fermentations. The discrepancies between the 5-l and 30-l fermentors with air supply were presumably due to the higher contribution of surface aeration over submerged aeration in the 5-l fermentor. This study, therefore, proved the positive effect of high-purity oxygen in enhancing viable cell density as well as target recombinant protein production in microbial fermentations.
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제안 방법
The effects of supplying high-purity oxygen on HSA synthesis, cell growth, and by-product formation were investigated in a 5-l as well as large-scale (30-l) fermentor. This study, using a vacuum pressure swing adsorption (VPSA) oxygen generator to supply enough oxygen for high cell density culture, showed a model of the highpurity oxygen fermentation process for mass production of recombinant proteins by large-scale high cell density cultures of recombinant H. polymorpha.
pH and temperature are key factors that should be optimized for improving cell growth and recombinant protein production of many cultivating microorganisms. Through several arrays of flask cultures to find the optimal condition for HSA expression, pH 6.0 and 37℃ were selected in this work. We also tested different expression systems, including constitutive expression using YPM medium and inducible expression using YPD or YPG media; the inducible system yielded about 1.
이론/모형
After fractionation, the gel was stained by Coomassie blue and destained by destaining solution (methanol 10%, acetic acid 10%). Protein yields were determined using PRO-MEASURE Protein Measurement Solution (Intron Biotech, Korea), which employs the calorimetric Bradford method [2]. The HSA concentration was measured by using the specific affinity of bromocresol green (BCG) to albumin.
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